This invention relates to novel chimeric human antibody light chain frameworks, comprising framework regions I to III from Vκ and framework region IV from Vλ, with advantageous properties, such as high stability and reduced aggregation propensity.
In the past forty years since the development of the first monoclonal antibodies (“mAbs”; Köhler & Milstein, Nature. 256 (1975) 495-7), antibodies have become an increasingly important class of biomolecules for research, diagnostic and therapeutic purposes. Initially, antibodies were exclusively obtained by immunizing animals with the corresponding antigen of interest. While antibodies of non-human origin can be used in research and diagnostics, in therapeutic approaches the human body may recognize non-human antibodies as foreign and raise an immune response against the non-human antibody drug substance, rendering it less or not effective. Thus, recombinant methods have been set up to render non-human antibodies less immunogenic.
Initial efforts to convert non-human mAbs into less immunogenic therapeutics entailed the engineering of chimeric antibodies consisting of animal (for example rodent) variable domains and human constant regions (Boulianne et al., Nature 312, (1984) 643-646). Further approaches aimed at the humanization of the rodent mAbs by introducing the CDRs in human variable domain scaffolds (Jones et al. Nature 321 (1986) 522-525; Riechmann et al., Nature 332 (1988) 323-7) or by resurfacing the variable domains (Roguska et al., Proc. Natl. Acad. Sci. USA 91 (1994) 969-973).
For the humanization by CDR loop grafting a human acceptor framework is either chosen based on homology to the donor framework (e.g. Roguska et al., Protein Engineering 9 (1996) 895-904; WO/2008/144757 (for rabbits)) or based on a preferred stability profile (Ewert et al., Methods 34 (2004) 184-199). The latter concept has been utilized for the humanization of rabbit antibodies onto a universal variable domain framework (U.S. Pat. No. 8,193,235).
With any chosen approach the resulting mAb or functional fragment ideally retains the desired pharmacodynamic properties of the donor mAb, while displaying drug-like biophysical properties and minimal immunogenicity. With respect to the biophysical properties of mAbs or functional fragments thereof, the propensity for aggregation has been a major concern for the developability of therapeutic molecules, mainly for the following three reasons:
First, protein aggregates generally show a higher potential to elicit an immune reaction in the host leading to the formation of anti-drug antibodies and eventually to drug neutralizing antibodies (Joubert et al., J. Biol. Chem. 287 (2012) 25266-25279).
Second, aggregates affect the manufacturing yield due to the increased effort for their removal (Cromwell et al., AAPS Journal 8 (2006), Article 66).
Third, off-target effects may be observed. The concern about oligomer formation is even more pronounced for applications where monovalent binding is preferred, including bispecific (or multi-specific) antibody formats with only one valency per target and construct, because oligomer formation in these cases results in protein conglomerates with multivalent binding properties potentially leading to off-target effects. An example for such unspecific activities is the use of a construct with a single CD3ε-binding domain in a bispecific antibody format. Such a format may for example bind with one of its two binding domains to a cancer antigen and with its second, CD3ε-binding domain recruiting cytotoxic T cells. Because cross-linking of the monovalent CD3ε-binding moiety is required to induce signaling through CD3ε, T cells will only be stimulated when engaged by multiple bispecific constructs bound to the surface of the target cell—and therefore adopting the properties of a cross-linked molecule—resulting in a specific T cell response that is exclusively directed towards the cancer cell. On the contrary, oligomers of such a construct would exhibit the properties of a cross-linked bispecific antibody and therefore activate cytotoxic T cells, even when not bound to cancer cells, thereby leading to systemic activation of T cells. Such unspecific and systemic activation of T cells could result is elevated cytokine levels leading to adverse effects.
Furthermore, a reliable and universally applicable acceptor framework is beneficial to enable a robust method of humanizing non-human antibodies, since cloning, expression and purification methods may be standardized.
To meet the above mentioned criteria for the humanization of non-human mAbs the published methodology proposes the use of human consensus variable domain framework sequences as acceptor scaffold for the engraftment of non-human complementarity determining regions. Based on the assumption that for each amino acid position in a protein, residues that contribute to protein stability have been enriched in the pool of germ line sequences during evolution, it is the common understanding that the closer the resulting humanized variable domains are to the human germ line consensus sequence of the respective variable domain family, the higher is the expected stability. This concept as described by Steipe (Steipe et al., J. Mol. Biol. 1994 240 (1994) 188-92) and reviewed by Wörn (Worn et al., J. Mol. Biol. 305 (2001) 989-1010) is widely accepted and finds wide-ranging application. Non-limiting examples are (a) the use of consensus sequence variable domains for the humanization of non-human antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289); (b) the use of consensus sequence variable domains to construct CDR libraries for in vitro screening of stable target-binding antibodies (Knappik et al., J. Mol. Biol. 296 (2000) 57-86); and (c) knowledge-based approaches to improve stability of antibody variable domains by exchanging non-consensus residues into consensus residues (Steipe, loc. cit.).
In addition, stabilities of the different variable domain families are described with VH3 being the most stable variable heavy domain. Importantly, in case of the variable light chain domains the Vκ family rather than the Vλ family is preferred (Ewert, loc. cit.). In particular, the human consensus sequences of VH3 and Vκ1 have been described as having favorable biophysical properties (Ewert, loc. cit.) and as being particularly suitable for the humanization of antibodies from non-human sources (use in Carter, loc. cit.).
In line with this there are several publications, in which the human Vκ1-VH3 consensus framework hu-4D5 has been used for the humanization of rodent and rabbit antibodies (Rader, J. Biol. Chem. 275 (2000) 13668-13676; WO/2005/016950; WO 2008/004834). Alternatively, a naturally occurring sequence belonging to the same families as hu-4D5 has been used to generate stable humanized single-chain (scFv) fragments from rabbit origin (U.S. Pat. No. 8,293,235; Borras et al., J. Biol. Chem. 285 (2010) 9054-9066).
Importantly it has to be noted that natural selection evolved stable variable domains always in the context of full-length antibodies, in which the variable domain is adjacent to and in contact with the constant domain 1. Therefore, it may well be that certain non-consensus residues provide better stability to the isolated variable domains, for example in the context of the single-chain Fv (scFv) fragment. In support with this hypothesis, non-consensus mutations contributing to stability have been described in US patent application US 2009/0074780.
Additionally, antibody stability is of crucial importance for production, purification, shelf-life and, as a consequence, the cost of goods for antibody therapeutics. Even minor improvements in one or more of these parameters may be highly relevant for the question of whether research and development of an antibody drug are going to be commercially viable.
Thus, despite that fact that many attempts have already been made to address the issue of obtaining humanized antibody drug substances from non-human antibodies, there still remains a large unmet need to develop novel human antibody frameworks with advantageous properties, such as high stability and reduced aggregation propensity, wherein the human antibody frameworks contain as few mutations as possible, ideally none, when compared to naturally occurring sequences, in order to reduce the risk of creating immunogenic sequences as far as possible. Such stable human frameworks could also be used to stabilize fully human antibodies or fragments thereof for example by loop grafting or simply by exchanging the stability-contributing component between the parent antibody and the stable framework.
The solution for this problem that has been provided by the present invention, i.e. novel chimeric human antibody light chain frameworks, comprising framework regions I to III from Vκ and framework region IV from Vλ, with advantageous properties, such as high stability and reduced aggregation propensity, has so far not been achieved or suggested by the prior art.